Introduction: An attractive treatment target following intracerebral hemorrhage (ICH) are reactive oxidative stress (ROS) reactions occurring after contact with blood breakdown products, such as hemin and iron. Recent work suggests the ferroptosis cell death pathway predominates after ICH, likely secondary to ROS and free iron consuming key protection pathways. While studies directed at iron with deferoxamine (DEF) are underway, DEF suffers from poor cellular uptake, short shelf life and toxicity. We report a new version of our well tolerated poly(ethylene glycol)-ated carbon nanodrug, PEG-HCCs, previously demonstrated to have enormous antioxidant capacity and beneficial effects in trauma and ischemia (Samuel et al, PNAS 2015), tested here in ICH models.
Methods: PEG-NH 2 and DEF have a terminal amine that can be covalently attached via an amide linkage to the HCC (Fig 1a) to achieve a loading of 25 DEF molecules per DEF-HCC-PEG. We tested the effectiveness of DEF-HCC-PEG against the consequences of hemin (5 M) and iron (50 M) in differentiated cultured SHSY-5Y cells and after SQ injection in mice following intracerebral hemolyzed blood. Doses used were based on prior work with PEG-HCCs.
Results: DEF-HCC-PEG enhanced DEF stability for at least 2 weeks, presumably benefiting from the antioxidant HCC parent. Immunofluorescence demonstrated rapid and robust cellular uptake. When treated .5-1 hour after hemin or iron, DEF-HCC-PEG showed excellent effects against double stranded (ds) DNA damage (p53BP1 and yH2A.X expression) and cell death. Mitochondrial action potential was restored at 24 hours (Fig 1b, c, d). Similar beneficial effects were seen in-vivo. For example, dsDNA damage was significantly reduced (Fig 1e).
Conclusions: We developed a novel nanodrug that addresses both key mechanisms of ferroptosis, ROS and excessive iron, while overcoming stability and uptake shortcomings of DEF alone. Studies assessing effect on functional outcome are underway.